Fret-based glucose-detection molecules

ABSTRACT

Proteins ( 20 ) having glucose-binding sites ( 28 ) that bind to glucose ( 30 ) are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application claims priority to U.S. provisionalpatent application 62/325,136 to Biron-Sorek et al., filed Apr. 20,2016, and entitled “FRET-BASED GLUCOSE-DETECTION MOLECULES,” which isincorporated herein by reference.

FIELD OF THE INVENTION

Some applications of the present invention relate generally to sensormolecules for detecting an analyte in a body. More specifically, someapplications of the present invention relate to sensor molecules thatprovide an optical signal that is indicative of detection of theanalyte.

BACKGROUND

The monitoring of various medical conditions often requires measuringthe levels of various components within the blood. In order to avoidinvasive repeated blood drawing, implantable sensors aimed at detectingvarious components of blood in the body have been developed. Morespecifically, in the field of endocrinology, in order to avoid repeated“finger-sticks” for drawing blood to assess the concentrations ofglucose in the blood in patients with diabetes mellitus, implantableglucose sensors have been discussed.

One method for sensing the concentration of an analyte such as glucoserelies on Forster Resonance Energy Transfer (FRET). FRET involves thetransfer of energy from an excited fluorophore (the donor) to anotherfluorophore (the acceptor) when the donor and acceptor are in closeproximity to each other, leading to light emission by the acceptor. (Fclarity and correctness, this FRET-based emission is not referred toherein as fluorescence.) Because of the high sensitivity of the FRETsignal to the relative proximity of the fluorophores it is often used inbiological research as a measurement tool. For example, theconcentration of an analyte such as glucose can be measured by creatinga fused sensor which includes two fluorophores and a third moiety whichhas specific binding site for the analyte. The conformational change ofthe fused sensor which results from the binding of the analyte changesthe distance between the fluorophores, affecting the FRET signal andthus enabling the measurement of the analyte concentration.

PCT Patent Application Publication WO 2006/006166 to Gross et al., whichis incorporated herein by reference, describes a protein which includesa glucose binding site, cyan fluorescent protein (CFP), and yellowfluorescent protein (YFP). The protein is configured such that bindingof glucose to the glucose binding site causes a reduction in a distancebetween the CFP and the YFP. Apparatus is described for detecting aconcentration of a substance in a subject, the apparatus comprising ahousing adapted to be implanted in the subject. The housing comprises aForster resonance energy transfer (FRET) measurement device and cellsgenetically engineered to produce, in situ, a FRET protein having a FRETcomplex comprising a fluorescent protein donor, a fluorescent proteinacceptor, and a binding site for the substance.

An alternative approach to glucose sensing has been discussed e.g. by YJ Heo et al., in “Towards Smart Tattoos: Implantable Biosensors forContinuous Glucose Monitoring,” Adv. Healthcare Mater. 2013 January;2(1):43-56 (Epub Nov. 26, 2012). Heo et al. provide a review of theefforts to develop analyte monitoring methods, which include placing afluorescent material sensitive to a target analyte, e.g., glucose, underthe skin and reading the optical signal through the skin, thus enablingmeasurement of the analyte.

In recent years, improved far-red fluorophores, having a significantportion of their emission spectrum above 650 nm, have been developed inorder to exploit optical properties of biological tissue and enablein-vivo deep imaging, including, e.g., TagRFP, mRuby, mRuby2, mPlum,FusionRed, mNeptune, mNeptune2.5, mCardinal, Katushka, mKate, mKate2,mRaspberry and others. The relative emission of these fluorophores at anoptical window above 650 nm is typically 10-50%, enablingsufficiently-effective detection through the skin. Additionally,infrared phytochromes such as iRFP, IFP1.4, and IFP2.0 have beendeveloped which further push the emission spectrum into the infrared;however, these phytochromes depend on the availability of biliverdin,possibly complicating their practical use. Red fluorophores mayeffectively be used in conjunction with shorter-wavelengths fluorophores(e.g., green) to create FRET couples that can be used to developdifferent types of biosensors, as shown for example by Lam et al.

SUMMARY OF THE INVENTION

FRET-based glucose-detection molecules are described. Each moleculeprovides a FRET-based signal that is indicative of glucoseconcentration, and is sensitive within physiologically-relevant rangesand temperatures.

There is therefore provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinhaving SEQ ID No. 1.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinhaving SEQ ID No. 2.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinhaving SEQ ID No. 3.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinhaving SEQ ID No. 4.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinhaving SEQ ID No. 5.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinhaving SEQ ID No. 6.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinincluding an amino acid chain, the amino acid chain including an aminoacid sequence greater than 98 percent identical to SEQ ID No. 9, andresidue 16 of SEQ ID No. 9 is disposed at the glucose-binding site, andis a hydrophilic amino acid.

In an application, the amino acid sequence is SEQ ID No. 9.

In an application, amino acid 16 of SEQ ID No. 9 is a polar amino acid.

In an application, amino acid 16 of SEQ ID No. 9 is an uncharged polaramino acid.

In an application, amino acid 16 of SEQ ID No. 9 is Gln.

In an application, amino acid 16 of SEQ ID No. 9 is Asn.

In an application, amino acid 16 of SEQ ID No. 9 is an amidic aminoacid.

In an application, amino acid 16 of SEQ ID No. 9 is Gln.

In an application, amino acid 16 of SEQ ID No. 9 is Asn.

In an application:

-   -   the amino acid sequence is a first amino acid sequence,    -   the amino acid chain further includes a second amino acid        sequence that is greater than 98 percent identical to SEQ ID No.        11, and    -   the first amino acid sequence is closer to an N-terminal end of        the protein than is the second amino acid sequence.

In an application, the N-terminal end of the second amino acid sequencebegins immediately after the C-terminal end of the first amino acidsequence.

In an application, the amino acid chain further includes a fluorophoreamino acid sequence that defines a fluorophore and is disposed betweenthe C-terminal end of the first amino acid sequence and the N-terminalend of the second amino acid sequence.

In an application, the fluorophore amino acid sequence is adonor-fluorophore amino acid sequence, and defines a donor fluorophore.

In an application, the amino acid chain further includes anacceptor-fluorophore amino acid sequence that defines an acceptorfluorophore, and:

-   -   the first amino acid sequence is between the donor-fluorophore        amino acid sequence and the acceptor-fluorophore amino acid        sequence, and    -   the acceptor fluorophore is excitable by the donor fluorophore        by Förster Resonance Energy Transfer (FRET).

In an application, the amino acid chain further includes a linkersequence that connects the acceptor-fluorophore amino acid sequence tothe first sequence, and has SEQ ID No. 8.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinincluding an amino acid chain, the amino acid chain including an aminoacid sequence greater than 98 percent identical to SEQ ID No. 9, andresidue 16 of SEQ ID No. 9 is disposed at the glucose-binding site, andis Val.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding domain, theprotein including an amino acid chain, the amino acid chain including,in order: SEQ ID No. 7; SEQ ID No. 9; SEQ ID No. 10; and SEQ ID No. 11.

In an application, the amino acid chain further includes a Val-Ser-Lyssequence before SEQ ID No. 7.

In an application, the amino acid chain further includes SEQ ID No. 8between SEQ ID No. 7 and SEQ ID No. 9.

In an application, the amino acid chain further includes a Ser-Lyssequence between SEQ ID No. 9 and SEQ ID No. 10.

In an application, the amino acid chain further includes a Met-Valsequence between SEQ ID No. 9 and the Ser-Lys sequence.

In an application, the amino acid chain further includes a Glu-Leusequence between SEQ ID No. 10 and SEQ ID No. 11.

In an application, the amino acid chain further includes a Tyr-Lyssequence between the Glu-Leu sequence and SEQ ID No. 11.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding domain, theprotein including an amino acid chain, the amino acid chain including,in order:

-   -   optionally, a Val-Ser-Lys sequence;    -   SEQ ID No. 7;    -   optionally, SEQ ID No. 8;    -   SEQ ID No. 9;    -   optionally, a Met-Val sequence;    -   optionally, a Ser-Lys sequence;    -   SEQ ID No. 10;    -   optionally, a Glu-Leu sequence;    -   optionally, a Tyr-Lys sequence; and    -   SEQ ID No. 11.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinincluding an amino acid chain, the amino acid chain including:

-   -   a donor fluorophore region that has an amino acid sequence that        defines a donor fluorophore;    -   an acceptor fluorophore region that has an amino acid sequence        that defines an acceptor fluorophore that is excitable by the        donor fluorophore by Förster Resonance Energy Transfer (FRET);    -   a glucose-binding region that has an amino acid sequence that at        least in part defines the glucose-binding site; and    -   a linker sequence having SEQ ID No. 8 that connects the acceptor        fluorophore region to the glucose-binding region.

In an application, the glucose-binding region has SEQ ID No. 9.

In an application, the linker sequence connects a C-terminal end of theamino acid sequence of the acceptor fluorophore region, to an N-terminalend of the amino acid sequence of the glucose-binding region.

In an application, the donor fluorophore is at least 98% identical toClover.

In an application, the acceptor fluorophore is at least 98% identical tomKate2.

In an application, the acceptor fluorophore is at least 98% identical tomNeptune2.5.

There is further provided, in accordance with an application of thepresent invention, a protein having a glucose-binding site, the proteinincluding:

-   -   a donor fluorophore region that has a donor-fluorophore amino        acid sequence that defines a donor fluorophore;    -   an acceptor fluorophore region that has an acceptor-fluorophore        amino acid sequence that defines an acceptor fluorophore        that (i) is excitable by the donor fluorophore by Förster        Resonance Energy Transfer (FRET), and (ii) has a peak emission        wavelength in the red-to-far-red spectrum;    -   a glucose-binding region that defines the glucose-binding site,        the glucose-binding region having a glucose-binding-region amino        acid sequence that includes SEQ ID No. 9; and:    -   the protein has an amino acid sequence in which the        glucose-binding-region amino acid sequence is disposed between        the donor-fluorophore amino acid sequence and the        acceptor-fluorophore amino acid sequence, and    -   the protein is configured to reduce a distance between the first        fluorophore region and the second fluorophore region in response        to binding of glucose to the glucose-binding site.

In an application, amino acid 16 of SEQ ID No. 9 is Val.

In an application, amino acid 16 of SEQ ID No. 9 is a polar amino acid.

In an application, amino acid 16 of SEQ ID No. 9 is an uncharged polaramino acid.

In an application, amino acid 16 of SEQ ID No. 9 is Gln.

In an application, amino acid 16 of SEQ ID No. 9 is Asn.

In an application, residue 16 of SEQ ID No. 9 is a hydrophilic aminoacid.

In an application, amino acid 16 SEQ ID No. 9 is an amidic amino acid.

In an application, amino acid 16 of SEQ ID No. 9 is Gln.

In an application, amino acid 16 of SEQ ID No. 9 is Asn.

The present invention will be more fully understood from the followingdetailed description of applications thereof, taken together with thedrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a generalized FRET-basedglucose-detection molecule that represents the FRET-basedglucose-detection molecules described herein, in accordance with someapplications of the invention;

FIG. 2 is a graph that illustrates, in a generalized manner, FRETbehavior of the generalized molecule (and of the molecules that itrepresents), in accordance with some applications of the invention;

FIG. 3 is a schematic illustration of a generalized amino acid chain ofthe generalized molecule, in accordance with some applications of theinvention;

FIG. 4 is a set of graphs which show, for some of the glucose-detectingmolecules described, a FRET:F ratio at different glucose concentrations,measured at 35 degrees C., in accordance with some applications of theinvention; and

FIGS. 5A-B are graphs showing function of some of the glucose-detectingmolecules at different temperatures, in accordance with someapplications of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Reference is made to FIG. 1, which is a schematic illustration of ageneralized FRET-based glucose-detection molecule 20 that represents theFRET-based glucose-detection molecules described herein, in accordancewith some applications of the invention. Molecule 20 (as may each of themolecules that it represents) may be used in glucose-detecting implants,e.g., implanted subcutaneously in a subject. Molecule 20 (as do each ofthe molecules that it represents) comprises a glucose-binding region 22,a donor fluorophore region 23 that comprises a donor fluorophore 24, andan acceptor fluorophore region 25 that comprises an acceptor fluorophore26. Molecule 20 (e.g., glucose-binding region 22) comprises aglucose-binding site 28. In the disassociated state of molecule 20(state A), fluorophores 24 and 26 are not in a proximity to each otherthat allows FRET. Binding of a glucose molecule 30 to glucose-bindingsite 28 results in a conformational change in molecule 20 to itsassociated state (state B), bringing fluorophores 24 and 26 into aproximity that allows FRET.

A substantial portion (e.g., at least 20 percent, e.g., at least 40percent, e.g., at least 80 percent) and/or the peak of the emissionspectrum of acceptor fluorophore 26 is in the red and/or far-redspectrum (e.g., has a wavelength above 620 nm, such as above 650 nm,e.g., 620-850 nm, such as 650-800 nm). This facilitates, for example,the use of the molecules described herein within a subcutaneous implant,and transcutaneous detection of emission from fluorophore 26, e.g., by askin-mounted detector. For example, the gene encoding any of theFRET-based glucose-detection molecules described herein may be insertedinto mammalian cells, e.g., human cells, such as into the AAVS1 locus onchromosome 19, and the cells housed within the subcutaneous implant,such that the cells express the molecule. For some applications, thecells are human Retinal Pigment Epithelial (RPE) cells. For suchapplications, the gene encoding the molecule typically further comprisesa nucleotide sequence that encodes a signal peptide that promotessecretion of the molecule, as is known in the art. (The signal peptideis typically cleaved during or after secretion such that it does notfeature in the mature molecule.) For some applications, the signalpeptide is SEQ ID No. 12, which is described in Barash et al. (BiochemBiophys Res Commun. 2002 Jun. 21; 294(4):835-42) and whose amino acidsequence is:

MWWRLWWLLLLLLLLWPMVW A 21

For some applications, the molecules described herein are used incombination with devices and techniques described in the followingreferences, which are incorporated herein by reference:

-   -   PCT application IL2015/051022 to Brill, which published as WO        2016/059635;    -   U.S. Pat. No. 7,951,357 to Gross et al.;    -   US Patent Application Publication 2010/0160749 to Gross et al.;    -   US Patent Application Publication 2010/0202966 to Gross et al.;    -   US Patent Application Publication 2011/0251471 to Gross et al.;    -   US Patent Application Publication 2012/0059232 to Gross et al.;    -   US Patent Application Publication 2013/0006069 to Gil et al.;    -   PCT Publication WO 2006/006166 to Gross et al.;    -   PCT Publication WO 2007/110867 to Gross et al.;    -   PCT Publication WO 2010/073249 to Gross et al.;    -   PCT Publication WO 2013/001532 to Gil et al.;    -   PCT Publication WO 2014/102743 to Brill et al.;    -   U.S. Provisional Patent Application 60/588,211, filed Jul. 14,        2004;    -   U.S. Provisional Patent Application 60/658,716, filed Mar. 3,        2005;    -   U.S. Provisional Patent Application 60/786,532, filed Mar. 27,        2006;    -   U.S. Provisional Patent Application 61/746,691, filed Dec. 28,        2012; and    -   U.S. Provisional Patent Application 61/944,936, filed Feb. 26,        2014.

Quantitative detection of FRET-based emission is typically performed(e.g., by a detector unit) by comparing (i) emission from the acceptorfluorophore in response to excitation of the donor fluorophore (i.e.,due to FRET), with (ii) emission from the acceptor fluorophore inresponse to direct excitation of the acceptor fluorophore (which isreferred to herein as fluorescence), which serves as a control, e.g.,for variations such as distance between the molecule and the detector.The donor fluorophore is excited using light of a particular wavelengthrange (e.g., 430-520 nm), and direct excitation of the acceptorfluorophore is achieved using light of a different wavelength (e.g.,530-620 nm). The detector may measure (i) and then (ii), or vice versa.The ratio between (i) and (ii) is referred to herein as the“FRET:Fluorescence ratio” (“FRET:F ratio”).

For some applications, acceptor fluorophore 26 is (or is based on, e.g.,is greater than 98 percent identical to) the “mKate2” fluorophore. Forsome applications, acceptor fluorophore 26 is (or is based on, e.g., isgreater than 98 percent identical to) the “mNeptune2.5” fluorophore.Typically, donor fluorophore 24 is (or is based on, e.g., is greaterthan 98 percent identical to) the “Clover” fluorophore.

Reference is also made to FIG. 2, which is a graph that illustrates, ina generalized manner, FRET behavior of molecule 20 (and of the moleculesthat it represents), in accordance with some applications of theinvention. Three important features of molecules suitable for FRET-basedglucose detection are:

-   -   (1) High contrast. That is, a large difference between the        FRET:F ratio in the disassociated state and the FRET:F ratio in        the associated state (referred to herein as “delta ratio”        (“dR”)). In FIG. 2, this is represented as the difference        between the highest FRET:F ratio and the lowest FRET:F ratio on        the curve.    -   (2) High sensitivity at physiologically-relevant glucose        concentrations when at physiologically-relevant temperatures. In        FIG. 2, this is represented by the glucose concentration at the        mid-point (which is the steepest part) of the curve. This        concentration is referred to herein as “Kd”.    -   (3) Consistency across physiologically-relevant temperatures.        That is, the FRET:F ratio at a particular glucose concentration        should change as little as possible across temperatures that the        molecule may experience.

For such a molecule that is to be used in a subcutaneous implant, thephysiologically-relevant temperatures are 32-38 degrees C., e.g., 34-36degrees C., such as 35 degrees C. It is hypothesized by the inventorsthat a Kd, at 35 degrees C., of 2-10 mM glucose (e.g., 3-9 mM) isadvantageous for such a molecule that is to be used in a subcutaneousimplant.

In order to obtain an optimal biosensor, the inventors generated 300different protein molecules in a bacterial expression system. Themolecules included different FRET pairs (donor and acceptorfluorophores), and different links (e.g., linker sequences) between thevarious portions of the molecules (e.g., between fluorophore amino acidsequences and glucose-binding-region amino acid sequences). Themolecules were evaluated for their suitability, e.g., by testing dR, Kd,and for some, consistency across physiologically-relevant temperatures.

As a result of the above experimental approach, the following FRET-basedglucose-detection molecules were identified by the inventors as usefulFRET-based glucose-detection molecules:

Molecule D274 is defined by SEQ ID No. 1, whose amino acid sequence isas follows:

VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILAT 60SFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVK 120IRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRS 180KKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHRADTRIGVTI 240YKYDDNQMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVD 300PAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAA 360NQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAK 420DKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGA 480LAGTVLNDANNQAKATFDLAKNLADSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGD 540ATNGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQER 600TISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADK 660QKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRD 720HMVLLEFVTAAGITHGMDELGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK 773

Molecule D277 is defined by SEQ ID No. 2, whose amino acid sequence isas follows:

VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILAT 60SFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVK 120IRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRS 180KKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGMDEADT 240RIGVTIYKYDDNQMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKAL 300AINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLI 360AKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMW 420DTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALA 480LVKSGALAGTVLNDANNQAKATFDLAKNLADSKGEELFTGVVPILVELDGDVNGHKFSVR 540GEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPE 600GYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNV 660YITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKD 720PNEKRDHMVLLEFVTAAGITHGMDELGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK 779

Molecule D278 is defined by SEQ ID No. 3, whose amino acid sequence isas follows:

VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILAT 60SFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVK 120IRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRS 180KKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGMDEADT 240RIGVTIYKYDDNQMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKAL 300AINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLI 360AKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMW 420DTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALA 480LVKSGALAGTVLNDANNQAKATFDLAKNLADSKGEELFTGVVPILVELDGDVNGHKFSVR 540GEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPE 600GYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNV 660YITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKD 720PNEKRDHMVLLEFVTAAGITHGMDGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK 777

Molecule D279 is defined by SEQ ID No. 4, whose amino acid sequence isas follows:

VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILAT 60SFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVK 120IRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRS 180KKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHRADTRIGVTI 240YKYDDNQMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVD 300PAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAA 360NQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAK 420DKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGA 480LAGTVLNDANNQAKATFDLAKNLADGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDAT 540NGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTI 600SFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQK 660NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHM 720VLLEFVTAAGITHGMDGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK 769

Molecule D137 is defined by SEQ ID No. 5, whose amino acid sequence isas follows:

VSKGEELIKENMHTKLYMEGTVNNHHFKCTHEGEGKPYEGTQTNRIKVVEGGPLPFAFDI 60LATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTVTQDTSLQDGCLIY 120NVKLRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRCDMALKLVGGGHLHCNLKTT 180YRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAVARYCDLPSKLGHKLNGMDE 240ADTRIGVTIYKYDDNAMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGV 300KALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQG 360DLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDT 420AMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPE 480ALALVKSGALAGTVLNDANNQAKATFDLAKNLADMVSKGEELFTGVVPILVELDGDVNGH 540KFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFK 600SAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNF 660NSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQS 720ALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGAADGTNWKIDNKVVRVPYVGVDKDNL 780 AEFSKK786

Molecule D138 is defined by SEQ ID No. 6, whose amino acid sequence isas follows:

VSKGEELIKENMHTKLYMEGTVNNHHFKCTHEGEGKPYEGTQTNRIKVVEGGPLPFAFDI 60LATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTVTQDTSLQDGCLIY 120NVKLRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRCDMALKLVGGGHLHCNLKTT 180YRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAVARYCDLPSKLGHKLNGMDE 240ADTRIGVTIYKYDDNAMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGV 300KALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQG 360DLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDT 420AMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPE 480ALALVKSGALAGTVLNDANNQAKATFDLAKNLADMVSKGEELFTGVVPILVELDGDVNGH 540KFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFK 600SAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNF 660NSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQS 720ALSKDPNEKRDHMVLLEFVTAAGITHGMDGAADGTNWKIDNKVVRVPYVGVDKDNLAEFS 780 KK 782

Reference is now made to FIG. 3, which is a schematic illustration of ageneralized amino acid chain 60 of generalized molecule 20, inaccordance with some applications of the invention. Amino acid chain 60is thereby a generalization of the amino acid chains of the FRET-basedglucose-detection molecules described hereinabove, in accordance withsome applications of the invention. It is to be understood that FIG. 3is intended to show certain features of the amino acid chain of each ofthe FRET-based glucose-detection molecules described hereinabove, inorder to schematically illustrate commonalities and differences betweenthe FRET-based glucose-detection molecules described herein. Inparticular, it is to be understood that FIG. 3 is intended to illustratepresence or absence of certain sequences, and/or the order in whichcertain sequences within the amino acid chains are disposed with respectto each other, and is not intended to represent the relative lengths ofthose sequences.

An acceptor-fluorophore amino acid sequence 66 defines acceptorfluorophore region 25 (and thereby acceptor fluorophore 26), and islocated near (e.g., at) the N-terminus of chain 60. Further along chain60 is a glucose-binding-region amino acid sequence 62 a, which definesglucose-binding site 28. Still further along chain 60 is adonor-fluorophore amino acid sequence 64, which defines donorfluorophore region 23 (and thereby donor fluorophore 24). Still furtheralong chain 60, near (e.g., at) the C-terminus of chain 60 is a secondglucose-binding-region amino acid sequence 62 b. Glucose-binding region22 is derived from E. coli mg1B (galactose binding protein), whosesequence is modified, inter alia by division into sequences 62 a and 62b, with sequence 66 therebetween. Although glucose-binding site 28 isdefined by sequence 62 a, glucose-binding region 22 as a whole may beconsidered to be defined by sequences 62 a and 62 b together. Sequences66, 62 a, 64, and 62 b are present in all of the molecules describedhereinabove, and in the order shown in FIG. 3.

Generalized amino acid chain 60 also comprises sequences 70, 72, 74, 76,78, and 80, which are each present in at least one of the moleculesdescribed hereinabove, in the order shown with respect to the othersequences that are present in the molecule.

Sequence 70 is present only in molecules D137 and D138, in which thesequence is at the N-terminal end of the molecule. Sequence 70 may alsodefine part of the acceptor fluorophore.

Sequence 72 is present only in molecules D137, D138, D277, and D278, inwhich the sequence connects sequence 66 to sequence 62 a.

Sequence 74 is present only in molecules D137 and D138, in which thesequence connects sequence 62 a to the subsequent sequence.

Sequence 76 is present only in molecules D274, D277, D278, D137, andD138, in which the sequence connects the previous sequence (sequence 62a, for D274, D277, and D278; sequence 74 for D137 and D138) to sequence64.

Sequence 78 is present only in molecules D137, D274, and D277, in whichthe sequence connects sequence 64 to the subsequent sequence (sequence62 b for D274 and D277; sequence 80 for D137).

Sequence 80 is present only in molecule D137, in which the sequenceconnects sequence 78 to sequence 62 b.

Therefore:

-   -   sequence 70 may be described as an N-terminal sequence that is        present in a subset of the molecules described hereinabove;    -   sequence 72 may be described as a linker sequence that links        sequences 66 and 62 a in a subset of the molecules described        hereinabove;    -   sequences 74 and 76 may be described as linker sequences that        link sequences 62 a and 64 in subsets of the molecules described        hereinabove; and    -   sequences 78 and 80 may be described as linker sequences that        link sequences 64 and 62 b in subsets of the molecules described        hereinabove.

Sequence 70 has the following amino acid sequence:

VSK 3

Typically, sequence 66 has SEQ ID No. 7, whose amino acid sequence is:

XXELIKENMHXKLYMEGTVNNHHFKCTXEGEGKPYEGTQTXRIKXVEGGPLPFAFDILAT 60XFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTXTQDTSLQDGCLIYNVK 120XRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRXDMALKLVGGGHLXCNLKTTYRS 180KKPAKNLKMPGVYXVDRRLERIKEADXETYVEQHEVAVARYCDLPSKLGHX 231

In SEQ ID No. 7, each X represents a residue that may be one or anotheramino acid, according to the following:

-   -   1—X can be G or V (both of which are aliphatic)    -   2—X can be E or S    -   11—X can be T or M    -   28—X can be H or S    -   41—X can be N or M    -   45—X can be V or A (both of which are aliphatic)    -   61—X can be C or S    -   104—X can be V or A (both of which are aliphatic)    -   121—X can be L or I (both of which are aliphatic)    -   158—X can be C or A    -   171—X can be H or I    -   194—X can be F or Y (both of which are aromatic)    -   207—X can be N or K    -   231—X can be K or R (both of which are basic)

Sequence 72 has SEQ ID No. 8, whose amino acid sequence is:

LNGMDE 6

As described hereinabove, sequence 72 may be described as a linkersequence that links sequences 66 and 62 a, i.e., that links acceptorfluorophore region 25 to glucose-binding region 22 (and thereby to therest of the molecule) in a manner that facilitates FRET-based glucosedetection functionality. It is hypothesized that sequence 72 may also beused in other FRET-based glucose-detection molecules, by linking otheracceptor fluorophore regions (i.e., regions that define otherfluorophores) to glucose-binding region 22.

Typically, sequence 62 a has SEQ ID No. 9, whose amino acid sequence is:

ADTRIGVTIYKYDDNXMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGV 60KALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQG 120DLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDT 180AMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPE 240ALALVKSGALAGTVLNDANNQAKATFDLAKNLAD 274

In SEQ ID No. 9, residue 16 (represented by an X) may be a hydrophilic,polar (e.g., uncharged polar), and/or amidic amino acid, such as Q(e.g., as for molecules D274, D277, D278, and D279), or N (e.g., asdescribed hereinbelow). For some applications, residue 16 is A (e.g., asfor molecules D137 and D138). Alternatively, and as describedhereinbelow, residue 16 may be V.

Sequence 74 has the following amino acid sequence:

MV 2

Sequence 76 has the following amino acid sequence:

SK 2

Sequence 64 has SEQ ID No. 10, whose amino acid sequence is:

GEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTT 60FGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRI 120ELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQ 180QNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLLEFVTAAGITHGMD 231

Sequence 78 has the following amino acid sequence:

EL 2

Sequence 80 has the following amino acid sequence:

YK 2

Sequence 62 b has SEQ ID No. 11, whose amino acid sequence is:

GAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK 33

There is therefore provided, in accordance with some applications of theinvention, a protein having a glucose-binding domain, the protein havingan amino acid chain, the amino acid chain comprising, in order: SEQ IDNo. 7; SEQ ID No. 9; SEQ ID No. 10; and SEQ ID No. 11. For some suchapplications:

-   -   the amino acid chain further comprises sequence 70 before SEQ ID        No. 7;    -   the amino acid chain further comprises SEQ ID No. 8 between SEQ        ID No. 7 and SEQ ID No. 9;    -   the amino acid chain further comprises sequence 76 between SEQ        ID No. 9 and SEQ ID No. 10 (and may further comprise sequence 74        between SEQ ID No. 9 and sequence 76); and/or    -   the amino acid chain further comprises sequence 78 between SEQ        ID No. 10 and SEQ ID No. 11 (and may further comprise sequence        80 between sequence 78 and SEQ ID No. 11).

In accordance with some applications of the invention, a protein isprovided having a glucose-binding domain, the protein having an aminoacid chain, the amino acid chain comprising, in order:

-   -   optionally, sequence 70;    -   SEQ ID No. 7;    -   optionally, SEQ ID No. 8;    -   SEQ ID No. 9;    -   optionally, sequence 74;    -   optionally, sequence 76;    -   SEQ ID No. 10;    -   optionally, sequence 78;    -   optionally, sequence 80; and    -   SEQ ID No. 11.

Reference is now made to FIG. 4, which is a set of graphs which show,for some of the molecules described hereinabove, the FRET:F ratio (yaxis) at different glucose concentrations, measured at 35 degrees C.

The measurements were performed on proteins that were extracted from E.coli (in which they were produced) and purified in 50 mM Tris (pH 7.6, 1mM CaCl_2, 100 mM NaCl_2). Measurements were performed using an Infinite(R) 200 plate reader or on a fluoroscopic microscope. FRET-basedemission was measured by applying excitation at 475 nm and detectingemission at 640-800 nm. Direct fluorescence of the acceptor fluorophorewas measured by applying excitation at 575 nm and detecting emission at640-800 nm. Curves were generated from the measured points (typically10) by a non-linear interpolation, and Kd and dR were extracted.

The data may be summarized as follows:

D137: Kd = 37.8 mM glucose dR = 46 percent D138: Kd = 79.7 mM glucose dR= 54 percent D274: Kd = 2.7 mM glucose dR = 47 percent D277: Kd = 7.2 mMglucose dR = 38 percent D278: Kd = 5.8 mM glucose dR = 37 percent D279:Kd = 9.9 mM glucose dR = 37 percent

As described hereinabove, sequences 62 a and 62 b are derived from E.coli mg1B (divided between sequence 62 a and sequence 62 b). mg1B isdescribed, inter alia, in Vyas N K et al. (Science. 1988 Dec. 2;242(4883):1290-5), and Deuschle K et al. (Protein Sci. 2005 September;14(9):2304-14), and is archived at The Universal Protein Resource(UniProt; http://www.uniprot.org/uniprot/) as POAEES. These referencesare incorporated herein by reference.

In mg1B, residues 16 and 183 are key residues of the glucose-bindingsite. In the FRET-based glucose detection molecules described herein,these residues correspond to residues 16 and 183, respectively, ofsequence 62 a (e.g., of SEQ ID No. 9). Throughout this patentapplication, unless stated otherwise, reference to “residue 16” refersto this residue 16 (either the residue 16 of sequence 62 a, or thecorresponding residue in mg1B).

In wild-type mg1B, residue 16 is F (Phe/Phenylalanine). In D137 andD138, residue 16 is A (Ala/Alanine), which has been previously described(e.g., Deuschle K et al). In D274, D277, D278, and D279, residue 16 is Q(Gln/Glutamine), which (i) unlike F or A, is hydrophilic, (ii) unlike For A, is polar (e.g., uncharged polar), and (iii) unlike F or A, isamidic. It is hypothesized by the inventors that the advantageousreduction in Kd between (i) D137 and D138, and (ii) D274, D277, D278,and D279 (which brings the Kd of these molecules into the desirablerange described hereinabove) is due to the substitution of thehydrophobic phenylalanine or alanine, with the hydrophilic, polar (e.g.,uncharged polar), and amidic glutamine.

There is therefore provided, a glucose-binding molecule comprising anamino acid chain that has a glucose-binding-region amino acid sequencehaving SEQ ID No. 9, in which residue 16 is glutamine.

Placement of N (Asn/Asparagine) at residue 16 of sequence 62 a was alsotested. In a similar FRET-based glucose-detection molecule (of whichmolecule 20 is also representative), the following variants of residue16 of sequence were performed at room temperature:

D198 (16 = A): Kd = 3.2 mM glucose dR = 29.1 percent D241 (16 = Q): Kd =0.3 mM glucose dR = 21.4 percent D267 (16 = N): Kd = 9.4 mM glucose dR =32.3 percent D263 (16 = V): Kd = 7.9 mM glucose dR = 31.7 percent

It is to be noted that because these tests were performed at roomtemperature, the results are not directly comparable with thosedescribed with reference to FIGS. 4 and 5, which derive from testsperformed at higher temperatures. Nonetheless, at least due to thedemonstrated functionality of molecule D267, there is provided aglucose-binding molecule comprising an amino acid chain that has aglucose-binding-region amino acid sequence having SEQ ID No. 9, in whichresidue 16 is a hydrophilic, polar (e.g., uncharged polar), and/oramidic amino acid.

The results from molecule D263 suggest that for some applications,residue 16 may be valine. Nonetheless, the inventors hypothesize thatthe presence of a hydrophilic, polar (e.g., uncharged polar), and/oramidic amino acid (e.g., glutamine) at residue 16 of sequence 62 a(i.e., of SEQ ID No. 9) makes molecules based on molecule 20particularly suitable for in vivo FRET-based glucose-detection.

Reference is made to FIGS. 5A-B, which are graphs showing function ofsome of the glucose-detecting molecules at different temperatures, inaccordance with some applications of the invention. dR and Kd weremeasured for D274, D277, D278 and D279 at different temperatures, asdescribed hereinabove, mutatis mutandis. FIG. 5A shows dR for moleculesD274, D277, D278 and D279 at different temperatures, and FIG. 5B showsKd for the same molecules at the same temperatures. Between 31 and 38degrees C., the respective dR of molecules D277, D278 and D279 remainedsomewhat stable, whereas the dR of molecule D274 increased withtemperature. Kd increased with temperature for all four molecules; thechange was greatest for D279, and smallest for D274.

The understanding of temperature-based changes in Kd and dR for aparticular FRET-based glucose detection molecule, and/or theidentification of molecules with relatively temperature-stable Kd and/ordR is hypothesized by the inventors to improve the accuracy ofFRET-based glucose-detection systems in which such molecules are used.

Reference is again made to FIGS. 1-5B. As described hereinabove, theFRET-based glucose-detection molecules described herein are configuredfor use in a glucose-detecting implant, e.g., that operates with anextracorporeal (e.g., skin-mounted) detector that detects light emittedfrom the acceptor fluorophore. It is to be noted that the scope of theinvention includes not only the protein sequences described herein, butalso (i) gene sequences that encode these protein sequences, (ii) cells(e.g., mammalian cells) containing such gene sequences, and (iii)implants containing these protein sequences or gene sequences, and/orcells containing these protein sequences or gene sequences.

Additionally, the present disclosure contemplates sequences having atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, sequence identity to a reference sequence,wherein the reference sequence may include, for example, any of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any of sequences 70,66, 72, 62 a, 74, 76, 64, 78, 80, or 62 b.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed hereinabove. Rather, the scope of the present inventionincludes both combinations and subcombinations of the various featuresdescribed hereinabove, as well as variations and modifications thereofthat are not in the prior art, which would occur to persons skilled inthe art upon reading the foregoing description.

1-6. (canceled)
 7. A protein having a glucose-binding site, the proteincomprising an amino acid chain, the amino acid chain comprising an aminoacid sequence greater than 98 percent identical to SEQ ID No. 9, whereinresidue 16 of SEQ ID No. 9 is disposed at the glucose-binding site, andis a hydrophilic amino acid.
 8. The protein according to claim 7,wherein the amino acid sequence is SEQ ID No.
 9. 9. The proteinaccording to claim 7, wherein amino acid 16 of SEQ ID No. 9 is a polaramino acid.
 10. The protein according to claim 9, wherein amino acid 16of SEQ ID No. 9 is an uncharged polar amino acid.
 11. The proteinaccording to claim 10, wherein amino acid 16 of SEQ ID No. 9 is Gln. 12.The protein according to claim 10, wherein amino acid 16 of SEQ ID No. 9is Asn.
 13. The protein according to claim 7, wherein amino acid 16 ofSEQ ID No. 9 is an amidic amino acid.
 14. The protein according to claim13, wherein amino acid 16 of SEQ ID No. 9 is Gln.
 15. The proteinaccording to claim 13, wherein amino acid 16 of SEQ ID No. 9 is Asn. 16.The protein according to claim 7, wherein: the amino acid sequence is afirst amino acid sequence, the amino acid chain further comprises asecond amino acid sequence that is greater than 98 percent identical toSEQ ID No. 11, and the first amino acid sequence is closer to anN-terminal end of the protein than is the second amino acid sequence.17. The protein according to claim 16, wherein the N-terminal end of thesecond amino acid sequence begins immediately after the C-terminal endof the first amino acid sequence.
 18. The protein according to claim 16,wherein the amino acid chain further comprises a fluorophore amino acidsequence that defines a fluorophore and is disposed between theC-terminal end of the first amino acid sequence and the N-terminal endof the second amino acid sequence.
 19. The protein according to claim18, wherein the fluorophore amino acid sequence is a donor-fluorophoreamino acid sequence, and defines a donor fluorophore.
 20. The proteinaccording to claim 19, wherein the amino acid chain further comprises anacceptor-fluorophore amino acid sequence that defines an acceptorfluorophore, wherein: the first amino acid sequence is between thedonor-fluorophore amino acid sequence and the acceptor-fluorophore aminoacid sequence, and the acceptor fluorophore is excitable by the donorfluorophore by Förster Resonance Energy Transfer (FRET).
 21. The proteinaccording to claim 20, wherein the amino acid chain further comprises alinker sequence that connects the acceptor-fluorophore amino acidsequence to the first sequence, and has SEQ ID No.
 8. 22. A proteinhaving a glucose-binding site, the protein comprising an amino acidchain, the amino acid chain comprising an amino acid sequence greaterthan 98 percent identical to SEQ ID No. 9, wherein residue 16 of SEQ IDNo. 9 is disposed at the glucose-binding site, and is Val.
 23. A proteinhaving a glucose-binding domain, the protein comprising an amino acidchain, the amino acid chain comprising, in order: SEQ ID No. 7; SEQ IDNo. 9; SEQ ID No. 10; and SEQ ID No.
 11. 24. The protein according toclaim 23, wherein the amino acid chain further comprises a Val-Ser-Lyssequence before SEQ ID No.
 7. 25. The protein according to claim 23,wherein the amino acid chain further comprises SEQ ID No. 8 between SEQID No. 7 and SEQ ID No.
 9. 26. The protein according to claim 23,wherein the amino acid chain further comprises a Ser-Lys sequencebetween SEQ ID No. 9 and SEQ ID No.
 10. 27. The protein according toclaim 26, wherein the amino acid chain further comprises a Met-Valsequence between SEQ ID No. 9 and the Ser-Lys sequence.
 28. The proteinaccording to claim 23, wherein the amino acid chain further comprises aGlu-Leu sequence between SEQ ID No. 10 and SEQ ID No.
 11. 29. Theprotein according to claim 28, wherein the amino acid chain furthercomprises a Tyr-Lys sequence between the Glu-Leu sequence and SEQ ID No.11. 30-46. (canceled)